Measuring and increasing protein stability

Cn, Pace

doi:10.1016/0167-7799(90)90146-o

Cited by 460 publications

(358 citation statements)

References 56 publications

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“…a n and a u are the slopes of the pre-and post-transition of the baselines. The Gibbs energy change of GdnHCl-induced unfolding in the absence of GdnHCl [∆G(H 2 O)] was determined by the linear extrapolation model assuming a two-state transition, according to the following equations (33).…”

Section: Methodsmentioning

confidence: 99%

Kinetically Robust Monomeric Protein from a Hyperthermophile

et al. 2004

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Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl-and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40°C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50°C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (∆G(H 2 O)) at 50°C was 43.6 kJ mol -1 . The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90°C h -1 was higher than at a scan rate of 5°C h -1 . The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k u and ln k r values depended linearly on the denaturant concentration between 10 and 50°C. The ∆G(H 2 O) value obtained from the rate constant in water using the two-state model at 50°C, 44.5 kJ mol -1 , was coincident with that from the equilibrium study, 43.6 kJ mol -1 , suggesting the two-state folding of TkRNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.Microorganisms can be classified into psychrophiles, mesophiles, thermophiles, or hyperthermophiles on the basis of the differences in their optimal growth temperatures. The stabilities of proteins from these microorganisms exhibit different properties. Proteins from hyperthermophiles generally reveal greater stability than those from any other microorganisms. Therefore, proteins from hyperthermophiles are expected to provide unique information about protein folding, stability, and function (1-7).The stability of proteins in solution is evaluated by the Gibbs energy changes (∆G) upon denaturation, when they are reversible under experimental conditions. The value of ∆G is obtained from equilibrium and kinetic experiments. Studies concerning the stability of proteins from hyperthermophiles have focused primarily on the equilibrium aspects (8-13). The results suggested that extremely high stability of these proteins can be achieved by increasing the number of ionic interactions and the extent of hydrophobic surface burial. Several reports have recently appeared in which the equilibrium...

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Section: Methodsmentioning

confidence: 99%

Kinetically Robust Monomeric Protein from a Hyperthermophile

et al. 2004

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“…Interestingly, 4 of the 11 mutants studied have a significantly low m value (Glu63Gln, Asp10Asn, Asp76Asn, and Asp93Asn), while the others have m values similar to that of wild-type CT AcP. It is widely accepted that the m value is linearly correlated to the change of solvent exposed surface area upon denaturation (36,37). A decreased m value can be found for structurally very similar proteins and is generally explained either by the presence of an intermediate significantly populated at equilibrium during the unfolding reaction or by an increased compactness of the denatured state (38).…”

Section: Conformational Stability Of Mutated Ct Acpmentioning

confidence: 96%

The Contribution of Acidic Residues to the Conformational Stability of Common-Type Acylphosphatase

Paoli

Taddei

Fiaschi

et al. 1999

Archives of Biochemistry and Biophysics

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Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, ⌬G(H 2 O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.

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“…The data were analyzed assuming the free energy of unfolding or refolding, ⌬G, to be linearly dependent on the urea concentration (denoted here by C), as described in detail previously (17), …”

Section: Methodsmentioning

confidence: 99%

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

Caldinelli

Iametti

Barbiroli

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme D-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the ␤-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal ␣-helix containing the SerLys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal ␤␣␤ flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.

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Measuring and increasing protein stability

Cited by 460 publications

References 56 publications

Kinetically Robust Monomeric Protein from a Hyperthermophile

Kinetically Robust Monomeric Protein from a Hyperthermophile

The Contribution of Acidic Residues to the Conformational Stability of Common-Type Acylphosphatase

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

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